During the past year, we have been focusing on two areas of research. 1. Study structural biology of the Rev response element (RRE); 2. Study the structure and conformation of adenine riboswitch; 3. develop a technology for selective labeling of RNA molecules. 1. Nuclear export of HIV-1 mRNA requires specific interaction and cooperative oligomerization of the viral protein Rev with the Rev response element (RRE) in viral RNA. The structure of the RRE has been studied for more than two decades but its three dimensional structure remains unsolved. Here we report an 20 Angstrom-resolution solution structure of the RRE based on small-angle X-ray scattering (SAXS) analysis. The RRE adopts an A-like structure in which the two legs position the two known Rev binding sites 55 Angstrom apart, matching the distance between the two RNA-binding motifs in the Rev dimer. Mutational and functional studies indicate that the mechanistic and topological role of the RRE is to position two tracks of Rev binding sites at an optimal distance for specific and cooperative interaction with the Rev proteins. 2. Riboswitches are functional messenger RNA that regulates gene expression through conformational changes. In addition, riboswitch tertiary structure is also dependent on cation concentration as the RNA backbone is highly negatively charged. X-ray solution scattering in both small-angle (SAXS) and wide-angle (WAXS) is making an increasing impact on the understanding of the linkage between structure, dynamics and function. SAXS data provides RNA size, weight and shape information, and WAXS data are sensitive to small conformation changes in solution and provides a means to characterize the RNA conformational space in solution. We show small-angle and wide-angle X-ray scattering (SAXS &WAXS) can be used to probe Adenine riboswitch aptamer domain conformations as a function of ligand and ion concentration by combining with computational approaches. Multiple folding states were characterized in atomic levels. Our results revealed that the binding pocket changed its conformation in response to ligand binding. Our results suggest that the distal loop-loop interaction of Adenine riboswitch aptamer restricts the conformational freedom of the three-way junction to promote ligand binding; the ligand binding also restricts the freedom of three-way junction to promote the distal loop-loop interaction under physiological conditions. These results provide an integrated view of hierarchical folding in atomic levels in an adenine riboswitch aptamer as a function of ligand and ion concentration. 3. My group has developed a new method for selective labeling of RNA (SLOR) at designated residue(s) and/or segment(s) of large RNAs using solid-phase multi-cycle enzymatic reactions. The potential applications of SLOR are broad and far reaching, due to a wide range of roles that RNA plays in biology. The followings are just examples of a few areas. General RNA biochemistry, biophysics and molecular biology. The fluorescent labeled RNA molecules can be used to study interaction between/within RNAs, and between RNA and DNA, RNA and proteins in vitro or within the cellular environment following microinjection. For example, selectively labeled RNA can be used to study riboswitch mechanisms in regulation of gene expression. The fluorescent residues can be incorporated at two strategically locations in the riboswitch using SLOR in order to monitor the switching event that is directly synchronized with the relative movement between the aptamer and expression platform domains using time-resolved single molecule Forster Resonance Energy Transfer (FRET) experiments. Probing such an event has not been possible because of lack of the specifically fluorescent labeled riboswitch RNA molecules. RNA structural biology. RNA molecules alone are almost impossible to crystallize for structure determination. NMR spectroscopy is an ideal method for structure determination of RNAs since it is a solution-state method and does not require crystallization. However, it is limited to only small RNAs, up to 50 residues, because of the extensive overlaps of chemical shift signals and short lifetimes of NMR signals. With SLOR, selectively labeled RNAs at designated residue(s) and/or segment(s) can be used for recording NMR signals, resulting in greatly simplified NMR spectra for straightforward interpretation. Moreover, one can selectively deuterate designated residues/segments and record the signals from the remaining residues. The signals from the remaining residues will have a much longer lifetime, resulting in significant enhancement of both resolution and sensitivity of NMR signals. This enhancement will make it possible to determine high-resolution structures of much larger RNAs using NMR spectroscopy: this will revolutionize RNA structural biology. The SLOR method will have an immediate impact to several collaborations between my group and several other groups within NCI (those collaborations are part of reasons that I developed SLOR). Currently, we have filed a provisional patent for the SLOR technology.